Display of Streptococcus iniae α-Enolase on the Surface of Virus-Like Particles (VLPs) of Nervous Necrosis Virus (NNV) Using SpyTag/SpyCatcher

Virus-like particle (VLP)-based vaccines are promising candidates for overcoming the safety problems of live vaccines and weak immunogenicity of subunit vaccines. VLPs can be used as a platform for the development of combined vaccines by expressing foreign antigens, and foreign antigens can be displayed on the surface of VLPs by conjugation. In the present study, to use nervous necrosis virus (NNV) VLPs as a delivery tool for Streptococcus iniae α-enolase by displaying on the VLP's surface, the split-intein (SpyTag/SpyCatcher) conjugation system was used. NNV capsid protein fused to SpyTag (Capsid-SpyTag) and S. iniae α-enolase fused to SpyCatcher (α-enolase-SpyCatcher) were recombinantly produced, then mixed in various ratios. A ratio of Capsid-SpyTag to α-enolase-SpyCatcher of 1 to 1.5 showed the highest coupling efficiency corresponding to 83-92% of coupled capsid protein dimer and 32-52% of coupled capsid protein monomer. In TEM observation, VLP of Capsid-SpyTag had a regular shape and size of about 40 nm, while VLP fused with α-enolase-SpyCatcher showed an irregular shape and size of about 40-50 nm in diameter. In preliminary immunization experiments, olive flounder (Paralichthys olivaceus) and zebrafish (Danio rerio) immunized with VLP fused with α-enolase-SpyCatcher showed the lowest cumulative mortality against S. iniae infection.